Characterization and regulation of human placental cytochrome P-450HP

细胞色素P-450 HP（CYP 4 B1）cDNA是从人胎盘cDNA文库中分离得到的，Yokotani等从其核酸序列中推导出了其氨基酸序列，但由于其蛋白质尚未被研究，因此其作为一种酶的功能尚不清楚。本研究对细胞色素P-450 HP的一级结构进行了分析，并尝试在酵母和大肠杆菌表达系统中表达P-450 HP cDNA，以表征细胞色素P-450 HP的性质.对细胞色素P-450 HP、P-450 ka-1（CYP 4A 6）和P-450 Cam（CYP 101）的氨基酸序列进行了比对，并根据P-450 Cam的6个底物识别位点（SRS）估计了P-450 HP的6个底物识别位点（SRS）。细胞色素P-450 HP的估计SRS 1位于（A.A.110-121）区域。我们已经证明P-450 ka-1的区域（A.A.58-123）对底物特异性起重要作用（未发表的数据）。细胞色素P-450 HP的这一区域的氨基酸序列与细胞色素P-450 HP的这一区域的氨基酸序列相比， 关于我们 OchromeP-450 ka-1（同源性为32%）。这一结果表明细胞色素P-450 HP具有与ω-羟基化不同的功能。利用大肠杆菌表达系统表达细胞色素P-450 HP cDNA。将编码疏水性氨基酸的cDNA从5 '端切去6 bp、72 bp和102 bp，以便于在大肠杆菌中表达。细胞色素P-450 HP表达载体转化的大肠杆菌细胞膜和可溶性组分中均未检测到细胞色素P-450.利用酵母表达系统表达细胞色素P-450 HP cDNA。用AAAAAA替换cDNA阅读序列，制备大鼠肝微粒体组分，测定P-450含量（<0.003nmol/mg）。表达的P-450 HP对各种底物均无催化活性。从人基因组文库中克隆了细胞色素P-450 HP基因组5 '端的4.8kb的侧翼区。在其他P-450基因组中确定为调控元件的共有序列在该序列中未发现。这一结果表明，细胞色素P-450 HP的表达受到未知机制的调节。少

Cytochrome P-450 HP (CYP4B1) cDNA was isolated from human placental cDNA library and the amino acid sequence was deduced from the nucleic acid sequence by Yokotani et al. The function of cytocrome P-450 HP as an enzyme is not known, because the protein, cytochrome P-450 HP,have not been investigated. In this study, the primary structure was analyzed, and expression of P-450 HP cDNA have been tried with yeast and E.coli expression systems to characterize the nature of cytochrome P-450 HP.1. Amino acid sequences of cytochrome P-450 HP,P-450ka-1 (CYP4A6) and P-450cam (CYP101) were aligned, and six substrate recognition sites (SRS) of P-450 HP were estimated from those of P-450 cam. Estimated SRS 1 of cytochrome P-450 HP was located in the region of (A.A.110-121). We had showed that the region (A.A.58-123) of P-450ka-1 plays an important role for substrate specificity (unpublished data). The amino acid sequence of this region of cytochrome P-450HP is very variable compared with that of cyt … More ochrome P-450ka-1 (homology is 32%). This result indicates that cytochrome P-450 HP has different functions from omega-hydroxylation.2. Expression of cytochrome P-450 HP cDNA with E.coli expression system was tried. 6bp, 72bp and 102bp from 5'terminal of cDNA which coded hydrophobic amino acids were trimmed to facilitate expression in E.coli. Cytochrome P-450 was not detected in membrane and soluble fractions of E.coli transformed with cytochrome P-450 HP expression vecters.3. Expression of cytochrome P-450 HP cDNA with yeast expression system was performed. The reader sequense of cDNA was replaced with AAAAAA.Microsome fraction of transformant was prepared, and P-450 content was determined (less than 0.003 nmol/mg). Expressed P-450 HP showed no catalytic activities toward various substrates.4.8 kb of 5'franking region of cytochrome P-450 HP genome was cloned from human genomic library. Consensus sequences determined in other P-450 genome as a regulatory element were not found in the sequence. This result indicates that expression of cytochrome P-450 HP was regulated by unknown mechanism. Less